CALVIN B. COULTER 317 



of the optimal point, however, agglutination appeared at all concen- 

 trations. As a result of over thirty series of determinations the opti- 

 mum for agglutination of normal red cells may be given as pH 4.75. 

 In the great majority of experiments the optimum corresponded quite 

 sharply with this figure. 



With sensitized cells, a greater variation was noted in individual 

 experiments, due possibly to varying degrees of sensitization, but 

 when the cells were still heavily sensitized after the final washing, the 

 optimum for agglutination occurred regularly near pH 5.3. The op- 

 timum for precipitation of serum globulin, in which fraction of the 

 serum the immune bodies are carried, is given as pH 5.2 by Rona and 

 Michaelis,^" and the isoelectric point of typhoid immune body^'^ as pH 

 5.4 so that the flocculation of sensitized cells seems to be connected 

 with that of the specific immune serum. 



The Chemical Significance of the Isoelectric Point. 



From the data given by the titrations above described, and carried 

 out for the determination either of the isoelectric point or the optimum 

 for agglutination, one may construct a curve giving the amounts of 

 HCl added, as ordinates, on the pH values attained, as abscissae. 

 A similar curve may be drawn for the addition of acid to 10 cc. of 

 saccharose solution alone. From these two curves a third may be 

 drawn (Fig. 4) with pH values again as abscissae, and as ordinates the 

 differences between the ordinates of the first two curves, or the 

 amounts of HCl required to bring a suspension of cells to a given H 

 concentration in excess of those necessary in the case of the saccharose 

 solution alone. There is evident a sharp inflection in this curve at 

 pH 4.7. This point is so close to the value found for the isoelectric 

 point that we may speak of it as the same. The protein of the cells 

 exists then on the acid side of the isoelectric point as a different chemi- 

 cal substance from that occurring on the alkaline side. Since a true 

 "puffer" action is manifest, it appears that the protein combines with 

 hydrogen ion. An adsorption of hydrogen ion can hardly be thought 

 of here as anything but a chemical combination. 



^2 Rona, P., and Michaelis, L., Biochem. Z., 1910, xxviii, 193. 



